Apparatus for microscopic observation of long-term culture of single cell

ABSTRACT

An apparatus is provided with a cell culture container having a cell culture region made of a hole formed on a substrate, a semi-permeable membrane covering a top plane of the cell culture region and a culture medium replacement part provided over the semi-permeable membrane, a mechanism for supplying a cell culture medium into the cell culture container, and a microscopic optical mechanism for enabling long-term observation of the cell within the cell culture region. This apparatus makes it possible to culture a cell group originating from a particular single cell, to perform culture and observation while identifying cells to be subjected to interaction during the process of culturing cells, and observe a difference in variation between the particular cell and other cells. There is also provided a mechanism which makes it possible to collect only a cell assuming a particular state and perform analysis or biochemical measurement of a gene of the cell, an expressed mRNA and the like.

TECHNICAL FIELD

The invention of the present application relates to an apparatus for themicroscopic observation of long-term culture of a single cell. Morespecifically, the invention relates to an apparatus for microscopicobservation of long-term culture of a cell, which enables the culturingof cells in units of one cell while observing the state of a particularcell microscopically, in the field of biotechnology that usesmicroorganisms and cells, as well as to a method of observation usingsuch an apparatus.

BACKGROUND ART

Conventionally, in the fields of biology, medicine and pharmacy, changesin the state of a cell and the response of a cell to a certain drug hasbeen observed on the assumption that the average value of a cell clusterrepresents the characteristic of one cell. However, in reality, cellcycles rarely synchronize with one another in a cell cluster and each ofthe cells expresses protein at a different cycle. Although synchronousculture methods have been developed in order to solve such problems,because the cultured cells do not originate from the exact same cell,there is a possibility that different protein expressions occur due tothe difference between the genes of the individual pre-cultured cells.Hence, when the response to certain stimuli are actually analyzed, it isdifficult to determine whether the fluctuation in the results are causedby the response fluctuation belonging generally to the cell responsemechanism or from the difference between cells (i.e., the difference ingenetic information between cells). In addition, since cell strains arenot cultured from one cell, for similar reasons it is difficult todetermine whether the reproducibility of response to stimuli fluctuatewith the difference between the genes of individual cells. Furthermore,there are two kinds of stimuli (signals) to a cell: one that is given bythe quantities of signal substances, nutrition and dissolved gasescontained in the solution surrounding the cell, and one that is given byphysical contact with other cells. Until recently, in the research fieldof biotechnology, observation of a cell was done by temporarilyextracting part of a cell group cultured in a large culture unit andsetting it in a microscope. Alternatively, microscopic observation wasperformed using a microscope enclosed in a temperature-controlledplastic container, which further contains a smaller container with meansto control carbon dioxide concentration and humidity. In relation tosuch a method, various methods have been proposed for maintaining thesolution conditions during cell culture by replacing the used mediumwith fresh medium. For example, in the method disclosed inJP-A-10-191961, a circulation pump operates to raise the level of theculture medium above the top edge of the substrate, or lower the levelof the culture medium below the bottom edge of the substrate, andmaintains a constant nutritional state by supplying fresh culture mediumwhen the level of the culture medium is low, and discharging the culturemedium when the level of the culture medium is high. Further,JP-A-8-172956 discloses a structure consisting of a culture containerinto which is inserted an insertion tube for introducing fresh culturemedium into the culture container, an extraction tube for dischargingculture medium from the culture container, and a gas tube which connectsthe gas phase of the culture container and the pump, each comprising afilter for preventing bacteria from entering the culture container,which can maintain the nutritional state of the culture container at aconstant level.

However, in spite of these proposals, a method of culturing cells whilecontrolling the solution conditions as well as physical contact betweenthe cells has not yet been known. In addition, a means for selecting oneparticular cell and culturing the single cell as a strain is not known.Furthermore, the art of controlling solution conditions and cell densityin a container, or the art of culturing and observing cells whileidentifying cells that interact with one another has not been known,either.

As is apparent from the foregoing description, in conventionaltechnology, cell strains do not have the exact same gene because cellculture is initiated from a cell group. Further, in conventionaltechnology, it is difficult to select particular cells and culture theselected cells while controlling the interaction or the density of thecells. Furthermore, in conventional technology, although attempts tomaintain the solution condition by replacing the culture medium is beingmade, it is difficult to rapidly change the environment of a particularcell that is being cultured and observe the response of that cell.

Therefore, the subject of the present invention is to solve theabove-described problems of the prior art, and to provide a noveltechnical means for enabling: the culture of a cell group originatingfrom a particular single cell; the culture and observation of cellswhile identifying cells that interact with them; and the observation ofthe difference between a cell to which a substance that interacts withthe cells, such as a signal substance, has been added, and other cells.The invention also aims to provide novel means which enables thecollection of a cell that assumes a particular state and the analysis orbiochemical measurement of a gene or an expression mRNA of the cell.

SUMMARY OF THE INVENTION

In order to solve the above-described problems, the present inventionprovides an apparatus for the microscopic observation of long-termculture of a single cell, which comprises: a cell culture containercomprising a cell culture region consisting of a hole created on asubstrate, a semi-permeable membrane covering the cell culture regionand a culture medium exchange region on top of the semi-permeablemembrane; a means for supplying liquid medium to the cell, culturecontainer; and an optical microscopic means for long-term microscopicobservation of a cell within the cell culture region.

In addition, the present invention provides various features of the formof such an apparatus. For example, in the above-described apparatus formicroscopic observation of long-term culture of a single cell, a smallculture container is set on the optical path of a microscopicobservation system; the interior of the container comprises a cellculture region made of a small hole for culturing a cell, an opticallytransparent semi-permeable membrane that is coarse enough to preventcells from passing through, which covers the top of the cell cultureregion to prevent the cell from coming out of the hole, and a culturemedium exchange region that allows the culture medium to circulate onthe top of the semi-permeable membrane. The cell culture region includesone or a plurality of small holes each having a width of approximatelyseveral μm to several hundred μm, and the apparatus has a means forguiding a particular cell to the hole. The apparatus also has a meansfor supplying nutrition and oxygen required for the growth of the cellin the cell culture region to the cell from the solution exchangingregion by diffusion from the solution circulation part, which alsoenables excrements or secretion to be eliminated; and also has a meansfor optically observing the cell. In addition, the apparatus has a meansfor controlling the number and type of cells in each hole of the cellculture region by a non-contact trapping technique such as opticaltweezers and a carrying passage formed between each hole.

The apparatus of the invention also has a means for controlling thesolution temperature inside the container by temperate control meanssuch as a Peltier element. Further, the feed tube for feeding culturemedium from a culture medium reservoir to the solution exchanging regionhas a degassing means, such as a degassing cell or a gas replacementcell, as well as a means for controlling the type and density of gasdissolved in the culture medium.

Further, the apparatus of the present invention has a means for guidingthe tip of a pipet or the like to the top of a particular hole andspraying a drug or the like to exert the influence of the drug on asingle cell in the particular hole via the semi-permeable membrane, anda means for extracting one particular cell from a particular holethrough the semi-permeable membrane by means of a pipet or the like, aswell as a means for introducing a filler or the like into a particularhole by means of a similar pipet or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing an example of the basic constructionof this invention;

FIG. 2 is a schematic view showing the arrangement of the single-cellculture region shown in FIG. 1;

FIG. 3 is a schematic view showing an A—A cross section of thesingle-cell culture region shown in FIG. 1;

FIG. 4 is a schematic view showing one example of a method of bonding asubstrate and a semi-permeable membrane;

FIG. 5 is a schematic view showing the manner of cell trapping in thecell culture region;

FIG. 6 is a schematic view showing one example of the structure of holeson a substrate surface;

FIG. 7 is a schematic view showing one example of the structure of holeson a substrate surface;

FIG. 8 is a schematic view showing the cross-sectional structures of theholes shown in FIG. 7 that have different sizes on a substrate surface;

FIG. 9 is a schematic view showing one example of the structures ofholes on a substrate surface;

FIG. 10 is a schematic view explaining means for carrying a cell byusing optical tweezers from one to another one of the holes shown inFIG. 9;

FIG. 11 is a schematic view showing one example of the structures ofholes on a substrate surface;

FIG. 12 is a schematic view showing one example of the structures ofholes on a substrate surface;

FIG. 13 is a schematic view showing one example of the construction of acell culture region;

FIG. 14 is a schematic view showing one example of the construction of acell culture region;

FIG. 15 is a schematic view showing one example of the structures ofholes on a substrate surface;

FIG. 16 is a schematic view showing one example of the structures ofholes on a substrate surface;

FIG. 17 is a view showing the result of observation of the growth speedand division length of each generation of E. coli;

FIG. 18 is a view showing the result of observation of the volumedependence of growth of E. coli; and

FIG. 19 is a view showing the result of observation of the difference indivision time between generations of E. coli and the difference indivision time between the initial numbers of bacteria during division ofE. coli. Incidentally, the reference numerals used in the drawings areas follows.

101, 111 light source 102, 111 filter 103, 114 condenser lens 104 gasdischarging valve 105 culture container 106 cell culture regionsubstrate 107 stage having temperature control function 108 laser lightsource 109 movable dichroic mirror. 112 dichroic mirror 113 mirror 115camera 116 image processing/analyzing and recording device 121 culturemedium supplying device 122 heater 123 dissolved gas replacement device124, 127 pump 125, 126 tube 128 waste solution reservoir 131 objectivelens 132 stage moving motor 201 gas discharging value 202 culturecontainer 203, 204 tube 301 culture container 301A solution exchangingregion 302, 303 tube 304 semi-permeable membrane 305 cell culture regionsubstrate 306 cell culture region 307 adhesive seal 401 semi-permeablemembrane 402 cell culture region substrate 403 avidin 404 biotin 405cell culture region 501 cell culture region substrate 502 cell cultureregion 502 cell 504 semi-permeable membrane 601 cell culture regionsubstrate 602 cell culture region 701 cell culture region substrate 702,703, 704, 705, 706 cell culture region 801 cell culture region substrate802, 804 cell culture region 803, 805 cell 901 cell culture regionsubstrate 902 cell culture region 903 groove 1001 cell culture regionsubstrate 1002 cell culture region 1003 groove 1004 cell 1005 opticaltweezers 1101 cell culture region substrate 1102 cell culture region1103 groove 1104 cell reservoir 1202 cell culture region substrate 1202sample introducing part 1203, 1205 cell trapping hole 1204, 1206 cellculturing hole 1207 groove 1208 cell observing hole 1301 culturecontainer 1302, 1303 tube 1304 semi-permeable membrane 1305 cell cultureregion substrate 1306 hole 1307 adhesive seal 1308 pipet 1311, 1314,1317 flow of culture medium 1312 membrane of mineral oil 1313 culturemedium 1315 solution level regulating part 1316 solution outlet 1401cell culture region substrate 1402 hole 1403 cell 1404 semi-permeablemembrane 1411 solution discharging pipet part 1412 solution suckingpipet part 1413 flow of pipet-discharged solution 1414 flow ofpipet-sucked solution 1501 cell culture region substrate 1502, 1505,1508 electrode 1503, 1506, 1509 hole 1504, 1507 groove 1510 movingdirection of cell 1511, 1512 cell 1601 cell culture region substrate1602, 1603 electrode 1604, 1605 ultrasonic vibrator 1611 direction ofelectric field 1612 direction of ultrasonic radiation pressure

BEST MODE FOR CARRYING OUT THE INVENTION

The invention of this application has features such as those describedabove, and embodiments of the invention will be described below.

First of all, it must be clearly pointed out that the term “one (single)cell” provided in the invention of this application must not beconstrued as limiting the number of cells to be handled at a time toonly one call. A plurality of cells may be cultured in a hole of a cellculture region, and a feature of the invention of this applicationresides in the fact that even during the culture of a plurality ofcells, it is possible to control and observe the culture process and thelike of culture of a single particular cell. The term “one (single)cell” means that fact.

The term “long term” must not be construed as an absolute criterion, andis a relative criterion corresponding to the kind of each individualcell. In addition, it must be understood that longer-term control andobservation of the culture process and the like can be realized than inany of the related art methods.

The invention of this application is based on the above-describedpremises.

FIG. 1 shows an example of the basic construction of an apparatus formicroscopic observation of long-term culture. Referring to FIG. 1, theapparatus for microscopic observation of long-term culture according tothe invention of this application is provided with a culture container105 constructed to culture a microorganism or a cell and to enablereplacement of its culture medium. The apparatus is also provided with aculture medium supplying and draining system which provides a culturemedium while adjusting the composition and the temperature of a culturemedium to be sent into the culture container 105, the kind and thedensity of gas and atmosphere, and the like, and an microscopicobservation optical system for observing a cell in the culture container105 with the lapse of time and recording the observed result on video orin a personal computer or the like.

More specifically, the culture container 105 in which to culture a cellis provided with a gas discharging valve 104 for discharging gases suchas air remaining in the container, whereby the culture container 105 isstructured to be filled with a culture medium. The size of the bottom ofthe culture container 105 is made to be a size suitable for microscopicobservation. In addition, the culture container 105 is placed on a stage107.

Referring to a culture medium supplying and draining part, the culturemedium supplied from a culture medium supplying device 121 having thefunction of supplying a plurality of kinds of culture media or culturemedia having different densities to the culture container 105 is firstadjusted in solution temperature by a heater 122, and is guided to adissolved gas replacement device 123 via a tube and dissolved gascomponents such as air are adjusted by the dissolved gas replacementdevice 123. Then, the culture medium is adjusted in flow rate by a pump124, and is sent to the culture container 105 via a tube 125.

The culture container 105 is provided with another tube 126, and thesolution inside the culture container 105 passes through the tube 126and is sent to a waste solution reservoir 128 by suction with a pump127. The pump 124 and the pump 127, during observation, perform supplyand drain of the culture medium of the culture container 105 at the sameflow rate, but when the gas discharging valve 104 is in a closed state,either one of the pump 124 and the pump 127 can be omitted. The wastesolution reservoir 128 is fitted with a heater so that the temperatureof the culture medium can be adjusted, and by sending air or the like toa culture medium reservoir through a tube by a pump, it is also possibleto bring the air contained in the culture medium into a saturated state.

The culture medium reservoir may be connected to the waste solutionreservoir 128 via the tube so that a valve can be opened and closed tocirculate the culture medium to a supplying device such as the culturemedium supplying device 121. In this case, a filter may also be disposedin the tube at a halfway position thereof so that extra components canbe removed from a waste solution.

The optical system with the basic construction shown in FIG. 1 iscapable of illuminating a sample in two opposite directions, both fromabove and below. Light emitted from an upper light source 101 isadjusted to a particular wavelength by a filter 102, and is condensed bya condenser lens 103 and irradiated onto the culture container 105. Theirradiated light is used as transmitted light for observation with anobjective lens 131. A transmitted light image of the interior of theculture container 105 is guided to a camera 115 by a mirror 113 andforms an image on the photosensitive surface of the camera. Accordingly,the material of the culture container 105 and the material of a cellculture region substrate 106 for actually culturing a cell at the bottomof the culture container is desirably an optically transparent material.Specifically, glass such as borosilicate glass or quartz glass, resinsor plastics such as polystyrene, or a solid substrate such as a siliconsubstrate is used. Particularly in the case where a silicon substrate isused, light of wavelength 900 nm or more is used for observation. Lightirradiated from a lower light source 110 is guided to the objective lens131 by a dichroic mirror 112 after having been wavelength-selected. Thelight is used as excitation light for observation of fluorescence in theinterior of the culture container 105. Fluorescent light emitted fromthe culture container 105 is again observed with the objective lens 131,and only fluorescent light and transmitted light from which theexcitation light has been cut by a filter 114 can be observed with thecamera 115. At this time, by adjusting the combination of the filters102, 111 and 114, it is possible to observe only transmitted light oronly fluorescent light with the camera 115, or it is also possible toobserve a transmitted light image and a fluorescent light image at thesame time. In an optical path, a mechanism is also provided whichintroduces laser light generated by a laser light source 108 into theobjective lens 131 through a movable dichroic mirror 109. In the casewhere this laser is used as optical tweezers, the focus position of thelaser in the culture container 105 can be moved by moving the movabledichroic mirror. Image data obtained by the camera is analyzed by animage processing and analyzing device 116, and on the basis of variousother analysis results such as the measured result of the temperature ofa temperature measuring instrument attached to the culture container105, it is possible to drive the movable dichroic mirror 109 as well asa stage moving motor 132 which causes the stage to move freely in theX-Y-Z directions in order to control the position of the stage having atemperature control function, on which the culture container 105 isplaced. Accordingly, the shape of a cell can be recognized, and afterrecognition, the cell can be tracked and invariably positioned in thecenter of the image, or the image can be focused onto a particular cellby adjusting the distance to the objective lens. Otherwise, the movabledichroic mirror 109 and the stage 107 having a temperature controlfunction, on which the culture container 105 is placed, can becontrolled at a predetermined period, and the stage moving motor 132 canbe driven at predetermined intervals.

FIG. 2 shows the arrangement of the culture container shown in FIG. 1.FIGS. 3 and 5 shot an A—A cross section of FIG. 2.

The culture container 202 shown in FIG. 2 is, similarly to theabove-described container, provided with a gas discharging valve 201, atube 203 for supplying a culture medium, and a tube 204 for discharginga waste solution, and a cell culture region substrate 205 similar to thecell culture region substrate 106 shown in FIG. 1 is provided at thebottom of the culture container 202.

The culture container 202 may be made of, for example, glass, but it isalso possible to use various kinds of optically transparent containersmade of a resin such as polypropylene or polystyrene, instead of glass.

In addition, it is possible to realize observation with near-infraredlight of wavelength 900 nm or more by using a solid substrate such as asilicon substrate.

The cross-sectional view of FIG. 3 shows a culture container for cellculture according to the invention of this application as well as aconstruction with which this culture container is provided.

The culture medium supplied from the culture medium supplying device 121shown in FIG. 1 described above is transferred into a solutionexchanging region 301A of a culture container 301 via the tube 302 shownin FIG. 3. Then, the fresh culture medium stored in this solutionexchanging region 301A is replaced with an old culture medium insidecell culture regions 306 via a semi-permeable membrane 304.

The cell culture regions 306 are made of a plurality of holes providedin a substrate 305. These holes are sealed at their tops by thesemi-permeable membrane 304. Accordingly, the structure of the cellculture regions 306 is such that the cells sealed in the holes 306cannot come out of these holes and unwanted microorganisms such asbacteria are prevented from entering from a culture medium part.

The size of each of these holes needs to be larger than the size of onecell. Accordingly, in general, in the case where a cell is to becultured, the size of each of the holes, although depending on the sizeof the cell, can be made, for example, 3 mm or less in opening diameterand 300 μm in depth. More preferably, in order to advantageously achievethe expected object of the invention of this application, the openingdiameter is set to from 1 μm to 1 mm, far more preferably, from 10 μm to50 μm, and the depth is set to 100 μm or less. This opening diameter anddepth may be appropriately adjusted according to the size and kind ofcell to be cultured.

Regarding the height of the solution exchanging region 301A of theculture container 301, it in desirable that h be larger than the depthof the holes, in view of the diffusion of the culture medium.

In addition, regarding the thickness of the cell culture regionsubstrate, it is necessary to use a thick substrate because an objectivelens of high numerical aperture is used in the case where microscopicobservation and optical trapping are performed with a 100-powerobjective lens. For example, in the case of a substrate made ofborosilicate glass, it is necessary to use a substrate of thickness 0.3mm or less.

The holes that constitute the cell culture regions 306 may be formed asa plurality of holes as described above, and in these holes, objectivecells are cultured.

A waste solution of the culture medium is drawn from the solutionexchanging region 301A through a tube 303. Since the holes of the cellculture regions 306 are very shallow, the replacement of the culturemedium is rapidly performed, and the old culture medium is dischargedthrough the tube 303.

The semi-permeable membrane 304 has micropores each having a sizethrough which a cell cannot pass and an outside bacterium or the likecannot enter. In the invention of this application, more specifically,it is preferable that the semi-permeable membrane 304 be 10,000 or morein molecular weight MW, 0.2 μm or less in pore size, and opticallytransparent.

Since the semi-permeable membrane 304, as described above, has a poresize through which a cell cannot pass, unwanted microorganisms do notenter from the solution exchanging region 301A of the culture container301 or cells do not flow into the solution exchanging region 301A fromthe holes of the cell culture regions 306.

The substrate 305 and the culture container 301, as shown in FIG. 3 byway of example, are made to adhere to each other by an adhesive seal 307such as a silicone seal, whereby the culture medium is prevented fromleaking from the solution exchanging region 301A. The substrate 305 issealed by the semi-permeable membrane 304 with no clearance formedtherebetween, except the tops of the holes of the cell culture regions306. The reason for this is to disable a cell from moving between oneand another of the holes in the case where the cell culture regions 306are made of a plurality of holes.

As means for adhesion between the substrate 305 and the semi-permeablemembrane 304, for example, a method using a bond between avidin andbiotin is effective. FIG. 4 is a schematic cross-sectional view showingthis bond. In the case where a cellulose membrane is used as asemi-permeable membrane 401 and glass is used as a substrate 402 of cellculture regions, the —OH group of the semi-permeable membrane 401 ispartly converted to a —CHO group, and this is —(CO)—NH-bonded to biotinmodified with an amino group. In this manner, the surface of thesemi-permeable membrane 401 is modified with biotin 404. On the otherhand, the surface of the glass substrate 402 is modified with an aminogroup by a silane coupling agent, and after that, the amino group ismade to react with biotin having a —CHO group, whereby the surface ofthe substrate 402 can be modified with biotin similarly to thesemi-permeable membrane. Then, avidin 403 is added to cause thesemi-permeable membrane 401 to adhere to the cell culture regionsubstrate 402 with a biotin-avidin bond.

In this manner, except the hole portion of a cell culture region 405,the biotin (404) disposed by bonding on the surface of one of thesemi-permeable membrane 401 and the substrate 402 is bonded to thebiotin (404) disposed by bonding on the surface of the other via theavidin 403, whereby a superior seal effect can be realized.

FIG. 5 is a schematic view showing the status of culture of cells 503 inthe holes of cell culture regions 502 provided in a substrate 501. Inaccordance with culture according to the invention of this application,even in the case where, for example, a 60-power objective lens is used,the cells 503 in the holes of the cell culture regions 502 can beobserved with a phase contrast microscope, a differential interferencemicroscope or a fluorescence microscope similarly to the case ofordinary preparations. Incidentally, in FIG. 5, there is also shown asemi-permeable membrane 504.

Although bowl-shaped holes are shown in FIG. 5, various other shapessuch as rectangles and polygons may also be used. As shown in FIG. 6 byway of example, holes which serve as cell culture regions may be ofuniform or approximately uniform size and can be arranged on a substrate601 as a plurality of cell culture regions 602 in the pattern of beingspaced at equal intervals. Otherwise, as shown in FIG. 7, cell cultureregions 702, 703, 704, 705 and 706 may be provided on the substrate 701as holes which are gradually varied in size. FIG. 8 is a schematic viewshowing the status of culture of cells 803 and 805 in the holes of cellculture regions 802 and 804 having different sizes. At this time, thenumber of cells in each of the holes is one, but the holes differ fromeach other in cell density which is obtained by dividing the number ofcells in a hole by the volume of the hole. In this manner, bycontrolling the volume of each hole, it is possible to observe variousreactions of the same number of cells for different densities.

It goes without saying that the arrangement pattern and number of holeswhich serve as cell culture regions, as well as the sizes and shapes ofthe holes may be suitably determined.

According to the invention of this application, for example, by changingthe size (diameter) of a hole of a cell culture region, it is possibleto change the extent of the mean free path of a cell which is anobjective target, or by changing the number of objective target cells tobe placed into holes of the same size (diameter), it is possible tochange the cell density of each of the holes. In addition, if the shapeof a hole for cell culture is changed, it is possible to observe theinfluence and effect of the shape on a cell.

According to the invention of this application, as shown in FIG. 9 byway of example, a plurality of holes serving as cell culture regions 902and grooves 903 interconnecting these holes and serving as thin passageseach of which allows one cell to narrowly pass therethrough may beprovided on the surface of a substrate 901. By providing the grooves 903serving as such passages, it is possible to measure, for example, themoving velocity, runnability and the like of cells. In addition, it ispossible to transfer a cell from each of the cell culture region holesto an adjacent one via a passage by using particle trapping means suchas optical tweezers. By using optical tweezers, it is possible toisolate or select cells, and it is possible to cause particular cells tointeract with one another in a hole, as well as it is possible tocontrol the number of cells in a hole.

FIG. 10 is a schematic plan view showing the manipulation of such cellmovement. Holes serving as cell culture regions 1002 and grooves 1003serving as passages for interconnecting these holes are provided in asubstrate 1001, and a cell 1004 can be moved through the groove 1003from a hole A to another hole B of the cell culture regions 1002 byoptical tweezers 1005 means. The optical tweezers 1005 means in thiscase is means which has heretofore been well known, and is means forirradiating a target cell with a beam of laser light to trap the celland enabling the cell to be moved in its trapped state with the movementof the beam of laser light.

According to the means of the optical tweezers 1005, as shown in FIG. 11by way of example, in a structure in which a hole of a cell cultureregion 1102 provided in a substrate 1101 and a hole of a cell reservoir1104 are made to communicate with each other by a groove 1103 whichserves as a passage, a particular cell can be carried from the hole ofthe cell reservoir 1104 to the hole of the cell culture region 1102 bythe optical tweezers, and conversely, a particular cell can also betransferred or discarded into the hole of the cell reservoir 1104.Otherwise, as shown in FIG. 12 by way of example, a single-cellpurification culture system can also be assembled on a cell cultureregion substrate 1201. In this case, first of all, cells are introducedinto a sample introducing part 1202, and one of these cells is movedalong a groove by the use of trapping means such as optical tweezers,and is guided to a cell culturing hole 1204. A cell trapping hole 1203is disposed in this groove at a halfway position thereof to prevent acell from swimming from 1202 into a hole 1204. Then, one cell which isin a particular state is again taken out of a cell group cultured andgrown from the one cell in the hole 1204, and this cell is similarlymoved by the use of the trapping means, and is guided to a secondculturing hole 1206. Similarly, a cell trapping hole 1205 is disposed ata halfway position. When the cell cultured in the hole 1206 grows toassume a certain predetermined state, cells are carried to a cellobserving hole 1208 through a cell carrying groove 1207, and observationor the like is performed.

FIG. 13 shows one example of a culture container different from theembodiment shown in FIG. 2. In this example, a pipet 1308 is introducedfrom the outside, and is inserted through a semi-permeable membrane 1304to selectively collect cells in a hole 1306. For this reason, a culturecontainer 1301 is made open at its top, and a mineral oil 1312 isapplied to cover the top surface of a culture medium 1313 with which thecontainer is filled, thereby preventing the penetration of unwantedmicroorganisms. The amount of the culture medium to be introducedthrough a tube 1302 in the direction of an arrow 1311 is made smallerthan the amount of solution to be sucked in the direction of an arrow1317, and by the use of a solution level regulating part 1315, when thissolution level becomes lower than a solution outlet 1316, air is suckedto stop the suction of solution, and when the solution level becomeshigher to close the outlet 1316, the culture medium is again sucked, sothat the solution level is held at a constant height. In this example,the solution level regulating part is installed separately from theculture container 105 so that ripples on a solution surface do notinfluence optical observation. The pipet 1308 can be used for sucking acell, but can also be used for injecting a filler to close a particularhole or groove or for introducing a particular cell into a cell cultureregion through a semi-permeable membrane. For example, the pipet 1308can be used in the case of introducing a particular sample into thesample introducing part 1202 of the single-cell purification culturesystem shown in FIG. 12. At this time, it is possible to experiment thecell introduced by the pipet without any problem of contamination, bymoving the cell along the groove toward the hole sealed with thesemi-permeable membrane, by trapping means such as optical tweezersbefore other cells or the like penetrate through a cut in thesemi-permeable membrane. In addition, in the embodiment, since cellswhich assume a particular state can be collected in units of one cellwith a pipet 1, gene polymorphism analysis, mRNA expression analysis andthe like can be also performed on this one cell.

FIG. 14 shows an embodiment in which a reagent for causing induction orthe like is introduced into a cell in a particular hole 1402. In thiscase, a pipet has a double structure, and discharges a solution throughan inside pipet 1411 and sucks a solution through an outside pipet 1412.Accordingly, the structure of the pipet is such that the solutiondischarged from the inside pipet 1411 is distributed in the vicinity ofonly its outlet and is prevented from leaking outwardly from the outsidepipet 1412, by suction from the outside pipet 1412. Accordingly, thispipet can be brought to the vicinity of a particular hole and apply anaction to only a particular cell.

Incidentally, owing to the movement of cells by trapping and movingmeans such as the above-described optical tweezers according to theinvention, it is possible to control the density of particular cells ina hole of a cell culture region, and it is also possible to implementidentification of cells which interact with one another, control of thetime period of interaction, and the like. However, the cell trapping andmoving means is not limited to the above-described optical tweezers, andmay also be, for example, means using ultrasonic waves or means usingelectric fields.

FIG. 15 shows one embodiment in which a plurality of electrodes 1502,1505 and 1508 are introduced in a cell culture region substrate 1501.Since a cell has a peculiar charge in a solution according to the stateof the surface thereof, when a positive charge is applied to anelectrode 1502, a cell 1511 having the opposite charge can be attractedto a hole 1503. By using this technique, cells corresponding todifferent strengths of negative charges are gathered in the hole 1503and holes 1506 and 1509, respectively. In addition, if negative chargesare applied to the electrodes 1505 and 1508, cells cannot move betweeneach of the holes.

FIG. 16 shows one example in which electrodes 1602 and 1603 are disposedon a cell culture region substrate 1601 and ultrasonic vibrators 1604and 1605 are disposed. In this example, an electric field 1611 and anultrasonic radiation pressure 1612 are used as non-contact forces formanipulating a cell. The electric field applies to a cell an externalforce corresponding to the surface charge of the cell, while theultrasonic radiation pressure exerts over the cell an external forcecorresponding to the size and hardness of the cell. It is desirable thatthe frequency of an ultrasonic wave to be used at this time be made afrequency of 1 MHz or more so that the generation of bubbles(cavitation) can be restrained. In this embodiment, specifically, theultrasonic radiation pressure and the electric field are respectivelymade to act in different directions perpendicular to each other, wherebycell distribution corresponding to the charge and size of each cell canbe two-dimensionally developed. In addition, in this example, anexternal force due to an electric field and an external force due to anultrasonic wave are combined on one substrate to perform separationaccording to the kinds of cells, but external forces due to an electricfield and an ultrasonic wave may also be independently used to transporta cell. Accordingly, it is possible to achieve cell handling similar tothat used with optical tweezers.

In the invention of this application, it is possible to provide meansfor measuring the number of cells in a hole serving as a cell cultureregion, and further, it is possible to provide a pipet which can beinserted into a hole of a cell culture region through a semi-permeablemembrane and collect a particular cell in the hole or inject or collecta reagent or a filler in the hole. It goes without saying that variousother detailed practical forms can be used without being limited to anyof the above-described forms.

For example, as described above, in the apparatus for microscopicobservation of long-term culture of a single cell according to thisapplication, it is possible to obtain, for example, the followingsuperior advantages.

(1) A particular cell can be isolated and observed for a long time.

(2) The kind and the temperature of a culture medium can be freelychanged during culture.

(3) The volume and the shape of a culture container can be freely set.

(4) The number of cells being cultured can be accurately set duringculture.

(5) Other unwanted microorganisms do not enter a container in whichcells are being cultured.

By using the above-described apparatus of this application, as shown inFIG. 17 by way of example, in an observation of E. coli growth, it hasbeen confirmed that there is no difference in growth speed and divisionlength between each generation and one E. coli bacterium divides intotwo when reaching a two-fold length. On the other hand, as shown in FIG.18, it has been confirmed that growth depends on the size, i.e., thevolume, of a hole of a cell culture region. In FIG. 18, “larger”indicates 2×10⁻⁷ ml, and “small” indicates 2×10⁻⁹ ml.

As shown in FIG. 19, there is a difference in division time betweengenerations and between the numbers of initial bacteria, and it has beenconfirmed that the first division takes time until the beginning ofdivision and one cell takes a long time until the beginning of divisioncompared to the case of a plurality of cells.

This feature can be realized exclusively by the apparatus and the methodaccording to the invention of this application which enables long-termculture and microscopic observation at the level of a single cell.

INDUSTRIAL APPLICABILITY

As described hereinabove in detail, according to the invention of thisapplication, by solving the problem of the related art, there isprovided novel technical means which makes it possible to culture a cellgroup originating from a particular single cell, to perform culture andobservation while identifying cells to be subjected to interactionduring the process of culturing cells, to spray a substance whichinteracts with cells, for example, a drug such as a signal substance,onto only a particular cell in a cell group which is being cultured sothat cells are cultured at a constant cell density, and observe adifference in variation between the particular cell and other cells.According to the invention of this application, there is also providednovel means which makes it possible to collect only a cell assuming aparticular state and perform analysis or biochemical measurement of agene of the cell, an expressed mRNA and the like.

1. An apparatus for enabling isolation, long-term culture andobservation of a cell, said apparatus comprising: a cell culturecontainer having a culture medium exchange region; a culture mediumsupplying device that supplies culture medium to said cell culturecontainer; a substrate coupled to said cell culture container; a firstcell culture region formed in said substrate, said first cell cultureregion comprising a first hole and being formed to hold one or morecells therein; a second cell culture region formed in said substrate,said second cell culture region comprising a second hole and beingformed to hold one or more cells therein; a semi-permeable membranearranged so as to cover said first cell culture region and said secondcell culture region; and a groove formed in said substrate, wherein saidgroove connects said first cell culture region to said second cellculture region, and serves as a thin passage for a cell such that thecell can pass between said first cell culture region and said secondcell culture region, and wherein said culture medium exchange region isdisposed above said semi-permeable membrane.
 2. The apparatus accordingto claim 1, further comprising: a first electrode disposed in a vicinityof said first cell culture region; and a second electrode disposed in avicinity of said second cell culture region; wherein, when a charge isapplied to said first electrode, a cell disposed within said second cellculture region moves from said second cell culture region to said firstcell culture region via said groove that connects said first cellculture region to said second cell culture region.
 3. A method for themicroscopic observation of long-term culture of a single cell, whichcomprises the use of the apparatus of claim
 2. 4. The apparatusaccording to claim 1, wherein each of said first cell culture region andsaid second cell culture region have a diameter larger than or equal to1 μm and smaller than or equal to 1 mm, and a depth less than or equalto 100 μm, and wherein said semi-permeable membrane has a molecularweight of 10,000 or more and a pore size of 0.2 μm or smaller.
 5. Amethod for the microscopic observation of long-term culture of a singlecell, which comprises the use of the apparatus of claim
 4. 6. Theapparatus according to claim 1, wherein said cell culture container ismade of an optically transparent material.
 7. A method for themicroscopic observation of long-term culture of a single cell, whichcomprises the use of the apparatus of claim
 6. 8. The apparatusaccording to claim 1, wherein said semi-permeable membrane is fixed to atop surface of said substrate by avidin-biotin bonding.
 9. A method forthe microscopic observation of long-term culture of a single cell, whichcomprises the use of the apparatus of claim
 8. 10. The apparatusaccording to claim 1, wherein said cell culture container furthercomprises a draining mechanism, so that the culture medium supplied tosaid culture medium exchange region from said culture medium supplyingdevice is exchanged with used culture medium in said first and secondcell culture regions through said semi-permeable membrane, wherein theused culture medium is discharged by said draining mechanism.
 11. Amethod for the microscopic observation of long-term culture of a singlecell, which comprises the use of the apparatus of claim
 10. 12. Theapparatus according to claim 1, further comprising a valve operable todischarge gas remaining in said cell culture container.
 13. A method forthe microscopic observation of long-term culture of a single cell, whichcomprises the use of the apparatus of claim
 12. 14. The apparatusaccording to claim 1, further comprising a mechanism for controlling atemperature of the culture medium.
 15. A method for the microscopicobservation of long-term culture of a single cell, which comprises theuse of the apparatus of claim
 14. 16. The apparatus according to claim1, wherein the cell can be trapped and moved between said first cellculture region and said second cell culture region by an opticaltweezers.
 17. A method for the microscopic observation of long-termculture of a single cell, which comprises the use of the apparatus ofclaim
 16. 18. The apparatus according to claim 1, wherein the cell canbe tapped and moved between said first cell culture region and saidsecond cell culture region by an ultrasonic wave.
 19. A method for themicroscopic observation of long-term culture of a single cell, whichcomprises the use of the apparatus of claim
 18. 20. The apparatusaccording to claim 1, wherein the cell can be trapped and moved betweensaid first cell culture region and said second cell culture region by anelectric field.
 21. A method for the microscopic observation oflong-term culture of a single cell, which comprises the use of theapparatus of claim
 20. 22. The apparatus according to claim 1, wherein apipet can be utilized for spraying reagents into said first cell cultureregion and said second cell culture region and for collecting thereagents.
 23. A method for the microscopic observation of long-termculture of a single cell, which comprises the use of the apparatus ofclaim
 22. 24. The apparatus according to claim 1, further comprising: anoptical microscope which enables long-term microscopic observation of acell within said first cell culture region or said second cell cultureregion; and a filter inserted into an optical path of said opticalmicroscope so as to enable fluorescence observation of the cell.
 25. Amethod for the microscopic observation of long-term culture of a singlecell, which comprises the use of the apparatus of claim
 24. 26. Theapparatus according to claim 1, further comprising: a means foracquiring image data; a means for recognizing a shape of a particularcell in the image data; and a means for controlling a position of astage and a focal length of an objective lens so as to maintain theparticular cell in a center of a visual field.
 27. A method for themicroscopic observation of long-term culture of a single cell, whichcomprises the use of the apparatus of claim
 26. 28. The apparatusaccording to claim 1, further comprising a means for measuring thenumber of cells in said first and second cell culture regions.
 29. Amethod for the microscopic observation of long-term culture of a singlecell, which comprises the use of the apparatus of claim
 28. 30. A methodfor the microscopic observation of long-term culture of a single cell,which comprises the use of the apparatus of claim 1.